This invention relates generally to electrodialysis methods for separation and purification of preferred end products and in particular to electrodialysis methods for the purification and recovery of gluconic acid derivatives, such as 2-keto-L-gulonic acid from solutions removed from fermentation reactors and in-vitro reactors.
A gluconic acid derivative, namely 2-keto-L-gulonic acid (hereinafter KLG), is a key intermediate in the production of valuable compounds including ascorbic acid (Vitamin C). However, to obtain high yields during the conversion of KLG to ascorbic acid, the KLG must be highly purified with a limited amount of impurities.
A convenient method for producing KLG is through a fermentation process. However, since most fermentation broths are maintained at neutral or near neutral pHs by the addition of basic substances, salts of KLG rather than KLG are produced. Furthermore, the fermentation broth also contains cells, neutrals and other undesirable materials. These additional components may interfere with the downstream chemistry used to convert KLG to ascorbic acid, therefore, the KLG must be isolated from the broth. Thus understood, any fermentation process for KLG must be integrated with an efficient recovery and purification process.
U.S. Pat. No. 5,747,306 discloses a method of separation using electrodialysis. The viable fermentation broth is maintained at a near neutral or basic pH of between 5 and 9 by the addition of basic substances such as sodium hydroxide, potassium hydroxide or ammonia. The broth is then passed through an electrodialysis tank which contains repeating cation and anion exchange membranes wherein the salts of KLG are removed from the broth. The viable fermentation broth containing neutrals, such as nutrients to insure survival of the microorganisms, is then recirculated into the fermentation system for reuse. However, the electrodialysis process produces a stream of purified KLG salt which gives lower yields during the conversion to ascorbic acid.
Additionally, the prior art process of converting organic sugars to the salt of KLG by using live and metabolically active microorganisms is complicated and demands constant vigilance to maintain a viable and active fermentation broth for the growth and/or metabolism of the microorganisms to ensure acceptable conversion of the substrate to the KLG salt.
To overcome the problems related with a high concentration of KLG salts during recovery of KLG from a fermentation medium, U.S. Pat. No. 4,990,441 discloses a method of acidifying the fermentation medium with sulfuric acid thereby precipitating the salt cation with the sulfate anion and protonating the KLG anion. However, it should be noted, that the medium containing salts of KLG also contains inorganic impurities such as phosphate and chloride anions which will also be converted to their corresponding acids with the addition of sulfuric acid. As a result, these inorganic acids can be concentrated during the KLG recovery processes, such as evaporative crystallization or direct drying and can cause acid catalyzed degradation of KLG. To rectify this problem the prior art contacts the medium containing the KLG and other impurities with a cation and anion exchange resin to remove any ionized impurities. But, neutral organics present in the fermentation broth which include simple and complex sugars may be unionized at the solution pH and thus not removed by passing the medium through cation and anion exchange resins. As such, the recovery of purified KLG is limited by the presence of these neutrals in the medium. The neutral sugars are concentrated during evaporative crystallization of KLG which causes increased viscosity of the mother liquor. As a result, multiple pass crystallization becomes difficult and KLG recovery is limited. Also, the neutrals interfere with direct drying which is a preferred method of recovery because of higher KLG yield with lower capital costs.
Accordingly, methods are needed for the concentration and purification of gluconic acid derivatives, such as KLG, which provide a higher recovery yield of KLG without the salts thereof, without contamination by inorganic impurities and neutral organics and/or without the need to maintain a fermentation medium for the growth and/or metabolism of a living and active culture of microorganisms.
For purposes of this invention, the terms and expressions below, appearing in the specification and claims, are intended to have the following meanings:
xe2x80x9cGluconic acid derivativexe2x80x9d is defined as an organic acid derived from gluconic acid including, but not limited to, 2,4, keto-D-gluconic acid, 2,5, diketo-D-gluconic acid, idonic acid, 2-keto-L-gulonic acid (KLG), vanillic acid and ascorbic acid.
xe2x80x9cFermentation reactorxe2x80x9d is defined as a classical fermentation reactor wherein live and viable microorganisms or cells such as bacteria are used for metabolizing carbohydrates.
xe2x80x9cFermentation medium xe2x80x9d is a medium or broth derived from a classical fermentation reactor.
xe2x80x9cIn vitro reactorxe2x80x9d is a reactor wherein enzymes being substantially free of non-living, non-viable and non-metabolizing cell structures, enzymes attached to non-living, non-viable and non-metabolizing cell structures or enzymes immobilized on a substrate have the ability to chemically oxidize and/or chemically reduce substrates or intermediates on the path to synthesizing gluconic acid derivatives.
xe2x80x9cIn vitro reactor mediumxe2x80x9d is a medium or solution derived from an in vitro reactor comprising at least a salt of a gluconic acid derivative and a coenzyme.
xe2x80x9cCoenzymexe2x80x9d is an organic molecule required for the catalytic functioning of an enzyme, such as nicotinamide, adenine dinucleotide, nicotinamide adenine dinucleotide phosphate and mixtures thereof
xe2x80x9cNeutralsxe2x80x9d are defined as sugars and/or compounds that are substantially unionized at the solution""s pH.
xe2x80x9cNon-viable fermentation mediumxe2x80x9d means the conditions in the fermentation broth or medium, such as pH, are such that the microorganisms used in the fermentation process are incapable of survival therein and the medium or broth may not be recirculated back into the fermentation tank for reuse without pretreatment.
xe2x80x9cNon-living mediumxe2x80x9d means a medium removed from a fermentation reactor or in-vitro reactor such as a non-viable fermentation medium or in-vitro reactor medium wherein no living cells or living microorganisms capable of growth and/or metabolic activity for producing gluconic acid derivatives and/or salts thereof are present.
It is the principal object of this invention to provide novel electrodialysis methods for the concentration and purification of gluconic acid derivatives from an acidified and/or non-viable fermentation medium or an in-vitro reactor medium.
It is another object of the present invention to provide electrodialysis purification methods that result in higher recovery of KLG with subsequent higher yields of ascorbic acid thereby realizing lower capital and operating costs in the production of ascorbic acid.
It is a further object of the present invention to provide an electrodialysis purification method that results in a recovered product that will not be subject to acid catalyzed degradation and can make multiple passes through an evaporation crystallization recovery process because of reduced viscosity of the mother liquid.
It is still further an object of the present invention to provide an electrodialysis purification method that concentrates the end product, such as KLG, to such a high level of purity that direct drying of KLG can be employed giving near quantitative recovery of same.
It is another object of the present invention to provide an electrodialysis purification method that allows the recovery of expensive coenzymes and/or enzymes used in an in vitro reactor system for chemically synthesizing the salts of a gluconic acid derivative such as KLG.
All of the above objects may be accomplished by an electrodialysis purification method comprising the following steps of:
a) providing a non-living medium, such as an acidified and/or non-viable fermentation medium or an in vitro reactor medium comprising at least a gluconic acid derivative and/or salt thereof; and
b) removing the gluconic acid derivative from the non-living medium by electrodialysis thereby providing a concentrated solution comprising at least the gluconic acid derivative.
It is an additional object of the present invention to provide processes for preparing purified and concentrated gluconic acid derivatives comprising the following steps of:
a) providing an acidified and/or non-viable fermentation medium comprising at least a gluconic acid derivative, an inorganic impurity and a neutral, wherein the gluconic acid derivative is substantially protonated;
b) removing the gluconic acid derivative and inorganic impurity from the acidified and/or non-viable fermentation medium by electrodialysis thereby providing a concentrated acidified aqueous solution comprising at least the gluconic acid derivative and inorganic impurity;
c) separating the inorganic impurity from the concentrated acidified aqueous solution thereby providing a purified and concentrated aqueous solution of the gluconic acid derivative; and
d) recovering the gluconic acid derivative from the purified and concentrated aqueous solution.
The step of separating the inorganic impurity from the concentrated acidified aqueous solution may be accomplished by several methods including, but not limited to, electrodialysis and anion exchange resins. This method is particularly favorable when the acidified and/or non-viable fermentation medium contains a substantial amount of gluconic acid derivative such as KLG in the free acid form.
It is still another object of the present invention to provide processes for preparing highly purified and concentrated KLG comprising the following steps of
a) providing an acidified and/or non-viable fermentation medium comprising at least KLG, an inorganic impurity and a neutral;
b) removing at least the KLG and inorganic impurity from the acidified and/or non-viable fermentation medium by electrodialysis thereby providing a concentrated acidified aqueous solution comprising at least the KLG and inorganic impurity, and a waste stream comprising a spent acidified and/or non-viable fermentation medium substantially depleted of KLG; and
c) separating the inorganic impurity from the concentrated acidified aqueous solution by electrodialysis thereby providing a purified and concentrated aqueous solution of KLG.
Recovery of the purified KLG may be accomplished by any recovery method well known in the art including evaporative crystallization or direct drying.
Step (b) of the electrodialysis process may be accomplished by a first electrodialysis stack comprising:
(i) an anode in an anolyte compartment, the anode in contact with an anolyte stream, a cathode in a cathode compartment, the cathode in contact with a catholyte stream, and an electrodialysis (hereinafter ED) membrane stack disposed between the anode and the cathode. The ED membrane stack comprises at least one feed compartment, at least one concentrate compartment and alternating anion and cation exchange membranes dispersed between the feed and the concentrate compartments. The anion exchange membranes must preferentially transport KLG anions and inorganic anions to the exclusion of neutrals in the acidified fermentation medium. The acidified and/or non-viable fermentation medium is introduced into the feed compartment and an aqueous solution containing an acid or salt is introduced into the concentrate compartment. A sufficient voltage is applied across the anode and cathode such that protons or other cations migrate across the cation exchange membrane into the concentrate compartment and KLG anions and inorganic anions are transported across the anion exchange membrane into the concentrate compartment wherein a concentrated acidified aqueous solution is collected comprising at least KLG and inorganic impurities. The acidified and/or non-viable fermentation medium is essentially depleted of KLG and removed from the system as a waste stream.
Step (c) of the electrodialysis process may be accomplished by a second electrodialysis stack comprising:
i) an anode in an anolyte compartment, the anode in contact with an anolyte stream, a cathode in a cathode compartment, the cathode in contact with a catholyte stream, and an ED membrane stack disposed between the anode and the cathode. The ED membrane stack comprises at least one feed compartment, at least one concentrate compartment and alternating anion and cation exchange membranes dispersed between the feed and the concentrate compartments. The anion exchange membranes must preferentially transport inorganic anions and to the exclusion of KLG anions in the concentrated acidified aqueous solution. The concentrated acidified aqueous solution is introduced into the feed compartment and a aqueous solution containing an acid or salt is introduced into the concentrate compartment. A sufficient voltage is applied across the anode and cathode such that protons or other cations migrate across the cation exchange membrane into the concentrate compartment and inorganic anions are transported across the anion exchange membrane into the concentrate compartment. The KLG remains in the feed stream thereby providing a purified and concentrated aqueous solution of KLG.
The acidified and/or non-viable fermentation medium of the present invention preferably has a pH of less than 4.5, and more preferably less than 3.5, and most preferably less than 2 wherein the gluconic acid derivative and specifically KLG is substantially protonated. Substantially protonated as used herein means the gluconic acid derivative is at least 80% protonated and preferably greater than 90% protonated.
In an alternative embodiment, step (c) for separating the inorganic impurities may be performed before step (b) in the above process.
In the first electrodialysis stack, KLG and inorganic impurities are removed from the acidified and/or non-viable fermentation medium wherein KLG anions, as well as inorganic anions if present, are transported across an anion exchange membrane. In this instance, the membrane rejects the passage of neutrals. Hereinafter, this first electrodialysis cell is referred to as xe2x80x9cKLG EDxe2x80x9d.
In the second electrodialysis stack, inorganic impurities such as acids or salts are separated from KLG by using anion exchange membranes which are permselective for inorganic anions but which do not transport KLG anions. The anion exchange membranes must have a very high resistance to KLG anion transport so that inorganic anions will be transported with minimal loss of KLG. Hereinafter, this second electrodialysis stack is referred to as xe2x80x9cDesalting ED.xe2x80x9d Desalting ED refers to the removal of inorganic acids as well as inorganic salts.
It has been discovered by the inventors that an in-vitro reactor provides a more efficient and cost effective method for producing KLG if the coenzymes used as redox cofactors in the chemical synthesis of gluconic acid derivatives can be reused by returning the coenzymes to the in vitro reactor. The advantages of using the in-vitro reactor which has no living and metabolically active cells or microorganisms in the in-vitro reactor medium are numerous including the fact that alternative metabolic pathways used by living cells are shut down. Thus understood, conversion of the substrates, such as carbohydrates and/or intermediate molecules to a preferred end product is predetermined. Therefore, the substrates and/or intermediate molecules are not wasted by being converted to unwanted byproducts. Instead, the most effecient pathway of conversion is used thereby producing higher yields from the substrates or intermediate molecules to the preferred gluconic acid derivative.
Using an in-vitro reactor, the cells, if any, containing oxidizing and reducing enzymes are dead and/or non-existent and conversion to a gluconic acid derivative salt is strictly by a chemical redox reaction. However, coenzymes, used as redox cofactors in the chemical synthesis of gluconic acid derivatives, are needed in the reactor medium and recovery of these coenzymes is essential because of the high cost of replacement. With this in mind, the inventors have discovered a method using electrodialysis to recover these expensive and valuable coenzymes for reuse or recirculation back into the reactor vessel.
Thus understood, it is yet another object of the present invention to disclose processes for preparing highly purified and concentrated KLG comprising the following steps of:
a) providing an in vitro reactor medium comprising at least a gluconic acid derivative anion, a metal counterion and a coenzyme; and
b) introducing the in vitro reactor medium to an electrodialysis cell comprising at least one bipolar membrane wherein the gluconic acid derivative anion is protonated and the metal counterion adds a hydroxide ion thereby providing at least a concentrated aqueous solution comprising a gluconic acid derivative and a stream comprising a metal hydroxide solution. In addition a separate stream comprising the coenzyme may be included in the process.
Several different bipolar membrane electrodialysis (ED) stacks may be used in this embodiment including a two or three compartment bipolar membrane electrodialysis stack. The two compartment stack comprises an anode in an anolyte compartment, the anode in contact with an anolyte stream, a cathode in a cathode compartment, the cathode in contact with a catholyte stream, and a two compartment bipolar membrane ED stack disposed between the anode and the cathode. The two compartment bipolar membrane ED stack comprises at least one anion exchange membrane, a bipolar membrane positioned on opposite sides of the anion exchange membrane and spaced sufficiently to provide at least one feed compartment and at least one concentrate compartment, the anode and cathode positioned on different ends of the cell connected to a power source for providing an electric current through the cell stack. The anion exchange membranes must preferentially transport gluconic acid derivative anions, such as KLG anions, and inorganic anions to the exclusion of neutrally charged molecules. The in-vitro reactor medium containing at least the salt of a gluconic acid derivative is introduced into the feed compartment. Water or an aqueous solution of a gluconic acid derivative or a salt thereof is introduced into the concentrate compartment. A voltage is applied across the anode and cathode sufficient to dissociate water to form a proton and hydroxide ion at the bipolar membranes and to transport the gluconic acid derivative anion across the anion exchange membrane into the concentrate compartment of the bipolar membrane ED stack. Gluconic acid derivative anions are transported across the anion exchange membrane while the passage of neutrals are prevented. The gluconic acid derivative anion is converted to its acid form once it has been transported into the concentrate or acid compartment by the addition of proton formed at a bipolar membrane. The cation originally associated with the gluconic acid derivative anion in the feed solution is converted to a base by addition of the hydroxide ion formed at the bipolar membrane. The net effect is the formation of concentrated and purified gluconic acid derivative, such as KLG and a base, for example caustic soda, from the splitting of the KLG Na salt.
In the two compartment configuration, the hydroxide ion which displaces the KLG anion may be neutralized by the addition of CO2 into the feed solution so that the pH in the feed compartment remains in a range where base catalyzed decomposition of remaining KLG does not occur. As a result, the products of the two-compartment configuration are a purified and concentrated solution of KLG and a solution comprising at least one or all of the following including a metal carbonate or bicarbonate, a metal hydroxide, and coenzymes.
The three compartment bipolar membrane ED stack is comprised of a feed or diluent compartment disposed between a base compartment and a concentrate compartment; the base compartment is separated from the feed compartment by a cation exchange membrane; and the concentrate compartment separated from the feed compartment by an anion exchange membrane. These three compartment units are bounded on the ends by bipolar membranes, which supply hydroxide ions to the base compartment and protons to the concentrate compartment. The three compartment bipolar membrane ED stack consists of at least one group of these three compartment units disposed between an anode and a cathode. In the three-compartment configuration, a hydroxide ion is introduced to the base compartment where it combines with a cation which migrates across the cation exchange membrane from the feed compartment. Therefore, the addition of a buffering agent such as CO2 to the base is not necessarily needed because the feed solution of a gluconic acid derivative, such as KLG is isolated from the base by the cation membrane. In the alternative, CO2 may be included if the preferred end product in the base solution is a carbonate or bicarbonate. Thus, the products of the three-compartment configuration are a purified and concentrated solution of KLG in the concentrate compartment, a solution of metal hydroxide from the base compartment and a stream containing at least a coenzyme that may be collected and reused or recirculated back into the reactor vessel. If there are any neutrals included in the feed solution, they remain therein.
Common to both the two and three compartment configurations is the anion exchange membrane, which must have a low resistance to gluconic acid derivative anion transport and should be selective for gluconic acid derivative anion transport over the transport of any neutrals that may be found in the feed solution.
A purified solution of a gluconic acid derivative, such as KLG is produced which is suitable for recovery by further processing via evaporative crystallization or other recovery techniques, such as direct drying including spray drying or thin film evaporation. If unwanted inorganic acids are removed, a higher recovery of KLG is expected because the potential for acid catalyzed decomposition of KLG upon concentration is removed. The separation of organic impurities such as neutrals from the KLG allows greater recovery of KLG because any increased viscosity that may be due to inclusion of neutrals is reduced during evaporation. Furthermore, KLG that has been purified by ED may be spray dried without suffering loss of yield during downstream conversion to ascorbic acid.